This invention relates to body fluid testing, and is particularly concerned with the separation of blood into various of its components, to enable a variety of tests to be performed, and to the separation of other body fluids, such as, bone marrow, sperm/seminal plasma, urine, and saliva.
In the field of haematology and related areas such as gene therapy and forensic pathology, and in other fields, such as the determination of sperm fertilising potential and cell separation from bone marrow, hitherto it has involved complex and expensive apparatus and procedures, and has required considerable operator skills to be separate correctly the components of the particular body fluid. This has been particularly so with blood, consequent on the need for careful preparation of a blood sample.
In other areas such as the testing or screening of a foetus for one or more of a number of possible abnormalities, it has hitherto predominantly been effected by invasive techniques frequently to the discomfort of the mother.
It is the object of the invention to provide equipment of notable simplicity and relatively low cost, with attendant procedures well within the capabilities of junior members of laboratory staff, to enable separation of a body fluid into various of its components.
According to a first aspect of the present invention, a body fluid separation means comprises at least one first chamber, a cooperating filter extending across the or each first chamber, at least one second chamber respectively to cooperate with the at least one first chamber, and one or both of said first and second chambers having a connection to vacuum.
According to a second aspect of the invention, a body fluid separation means comprises a chamber, a filter extending across the chamber, a removable closure means at each end of the chamber and a connection means to connect the chamber to vacuum positioned to one or both sides of the filter.
According to a third aspect of the invention, a body fluid separation means comprises a first plate formed with two or more collection chambers, a second plate with a corresponding number of waste receiving chambers, a connection to vacuum for each said waste receiving chamber, a filter means between each collection chamber and cooperating waste receiving chamber, and a third plate forming a vacuum chamber to connect each waste receiving chamber to vacuum.
With the second aspect of the invention, the removable closure means may simply be a cap fitted to and in sealing engagement with the end of the chamber, and a second cap may be provided to be fitted to the opposite end of the chamber.
With the third aspect of the invention, the first plate, second plate, and third plate, are preferably detachably secured in sealing engagement, with the filters trapped between the first and second plates.
Insofar as each usage of the invention in any of its aspects is concerned, the filter extending across one end of the chamber, bridging a chamber, or trapped between the first and second plates is selected to suit the body fluid to be tested, and if required, is pre-treated in a manner making it suitably for use with the body fluid to be tested. Thus, in one form of construction it is preferred that a leukocyte trapping membrane filter is provided. Where it is a case of the separation of cells, a replaceable filter is provided of a character that permits the passage of, for example, maternal blood cells and the entrapment of, for example, foetal blood cells. In the case of sperm testing, the filter will enable the passage of seminal fluid whilst trapping the sperm. Similarly with bone marrow, the filter is selected to suit the entrapment of bone marrow cells whilst allowing the passage of plasma.
The material of the chamber and of the or each end cap, and the material of each plate, may be such that the chamber and the cap(s) and the plates, can be sterilised, such as by, for example, gamma radiation. The filter is preferably a disposable component, formed from a relatively inexpensive plastics material, to provide a body fluid separation means for one use only and then to be disposed of.
According to a fourth aspect of the invention, a method of separating body fluid into selective component parts comprises applying a sample of body fluid to one side of a filter, applying vacuum to the opposite side of the filter to draw body fluid through the filter and to leave the specified component trapped on the filter, and releasing the specified component from the filter either in situ or following removal of the filter.
Thus, with particular reference to the separating of the DNA content of blood, and for which the invention in its second aspect is particularly suited, a whole blood sample is applied to one side of a filter, vacumm is applied to the opposite side of the filter to draw the whole blood sample therethrough and separate the plasma and red cell content of the blood from the leukocyte cell content, leaving the leukocyte content trapped in the filter, followed by the application of water or, preferably, isotonic saline, to the same side of the filter to be drawn therethrough by the vacuum applied to the opposite side of the filter to lyse the leukocyte cells trapped in the filter. As an alternative to the application of water/isotonic saline, a chemical lysing agent or compatible cell detergent can be used to separate the plasma and red cell content of the blood from the leukocyte cell content, and to leave the leukocyte content trapped in the filter. Following lysing the, such as, removable end cap on the chamber now containing what is waste material can be removed and discarded, the chamber inverted and isotonic saline applied to the opposite side of the filter to wash out the cell contents of the leukocyte cells from the filter into an appropriate receptacle from where the DNA content is removed. The suitable receptacle may be the removable end cap on the other end of the chamber. Alternatively, following removal of the end cap containing waste material, it can be replaced by the end cap from the opposite side of the chamber, and isotonic saline can be applied to the same side of the filter to wash out the cell contents of the leukocyte cells into the clean end cap, from where the DNA content can be removed. As a still further alternative, it is possible for certain tests to leave the leukocyte content trapped in the filter and to serve as a test base for a variety of medical tests.
In the circumstance where it is the removal of leukocyte cells, it is preferred that the commercially available filter known in trade as Pall LK4 is used. It is also preferred that the end cap to receive the cell contents is provided with a suitable membrane to isolate the DNA content of the cells from other cell debris washed from the filter to provide a clean DNA sample able to be liberated from the membrane utilising conventional transfer techniques. The invention, in its application to DNA harvesting and leukocyte testing, by its equipment and its method avoids completely the need for any preparation of a blood sample, and allows the separation, collection, and gathering of the DNA content in exceedingly simple and efficient manner.
Whole blood may simply be applied to the filter through the open end of the chamber, without pre-preparation, and vacuum applied to the opposite side such as by a relatively simple laboratory vacuum pump, or by, for example, a standard medical syringe. This causes red cells and plasma to be drawn through the filter and collected in a removable end cap, leaving leukocytes trapped in the filter. With continued application of vacuum, water, isotonic saline, or a chemical lysing agent, or a compatible cell detergent is then applied to and drawn through the filter, causing chemical lysing, or lysing of the leukocytes by osmotic shock.
Once the cell contents have been collected, the DNA component can then be gathered by conventional techniques.
In other spheres of testing such as, for example, foetal testing, sperm counting, bone marrow testing, urine, and saliva testing, it is desirable to provide multiple samples, and for which the invention in its third aspect is particularly suited. Here, a sample is applied to each of the number of filters overlying a respective waste collection chamber on the second plate following which the first plate is applied to the second plate to trap the filters and the assembly of first and second plates applied to the third vacuum forming plate.
Thus, in the case of the screening and isolation of foetal trophoblasts (immature blood cells) from maternal blood a filter that allows the passage of maternal blood cells and which entraps foetal blood cells is employed, for example, a Pall J100 or similar membrane, with the gel-coated side positioned to receive the blood sample. The filter is further coated to render it non-stick, as is then sample collection chamber. A whole blood sample taken from a mother need not be the subject of any prior preparation and can be applied to each of the number of filters. With vacuum applied via the vacuum chamber to the waste collection chamber maternal blood cells of the blood are drawn through the filter and collected in the waste collection chamber, whilst foetal blood cells are left entrapped in the filter. The second and third plates are removed and the waste material in the waste collection chambers discarded, and the first plate inverted. Here again, it is preferred that the foetal cells are released from the filter into the collection chambers from where they can be removed for test. It is, however, possible that the foetal filters can be stripped of debris to leave the cells trapped in the filter and tests effected on the cells whilst on the filter.
In its preferred form, a procedure for isolating and screening foetal trophoblasts comprises isolating foetal blood cells from a maternal blood sample using a specially prepared membrane which allows the maternal blood cells to pass through the membrane leaving the foetal blood cells trapped within the filter, the foetal blood cells then being lysed and a chemical and specific antibody marker compatible with specific chromosomes added, the marker having the ability to flouresce under shortwave ultra violet light to signal an abormality in the foetal cells. Thus, as in the example of Downs Syndrome, chromosome 21 will be shown to be present in triplicate, instead of an duplicate. The chromosome 21 will appear on the screen under UV light as three flourescent dots.
It is envisaged that this non-invasive screen could be carried out from 10 weeks gestation. The technique can be used for any other genetically carried syndromes i.e. Huntingdons chorea, cystic fibrosis, and spina bifida.